Since the advent of the polymerase chain reaction (PCR) it was recognized that amplification can be used to estimate the initial concentration of a template nucleic acid. However, variations in the efficiency or other aspects of an amplification can and do result from a number of different influences on the reactions. Only if such variations are accounted for or neutralized can one hope to obtain quantitative results. A number of approaches have been used or proposed to address this fundamental issue for quantitative PCR. Multiplex quantitative PCR, or quantitative PCR in which a plurality of target nucleic acids is amplified and quantified in one reaction mixture presents additional challenges.